G6845-90001 QQQ Familiarizsation

Agilent MassHunter Workstation
Software Data Acquisition for

7000 Series Triple Quad GC/MS
Familiarization Guide
Before you begin 3
Prepare your system 3
Prepare the samples required for data acquisition 3
Exercise Develop an acquisition method for the 7000 Series 5
Task 1. Set the inlet and injection parameters 5
Task 2. Enter GC acquisition parameters 7
Task 3. Create acquisition method for finding precursor ions 11
Task 4. Acquire precursor ion scan data (Optional) 13
Task 5. Determine precursor ion masses 15
Task 6. Create acquisition methods for finding product ion masses 18
Task 7. Acquire scan data for finding product ions (Optional) 21
Task 8. Determine the product ions 23
Task 9. Create an MRM method 26
Exercise Create a quantitative analysis method 28
Task 10. Acquire MRM data (Optional) 28
Task 11. Create a quantitative analysis batch 31
Task 12. Create an MRM quantitative analysis method 33
Task 13. Quantitate a batch of unknown samples 42
This guide shows how to use the Agilent 7000 Series Triple Quad GC/MS
to acquire and analyze sample data. If you want to skip the data acquisi-
tion steps in this guide, use the demo data files located in the Benzodiaze-
pine directory shipped with the system (in the Benzodiazepine\Data
folder of your Data Acquisition installation disk).
Agilent Technologies
In this guide, you learn how to determine the best acquisition settings for
analyzing your compounds of interest. These instructions help you understand not
only how to set up a method to optimize instrument parameters for best sensitivity
in acquisition, but also how to use the Qualitative Analysis program to identify
parameter values producing optimum signal response. You can also learn about
the Qualitative Analysis program by using the Qualitative Analysis
Familiarization Guide and the Quantitative Analysis program by using the
Quantitative Analysis Familiarization Guide.
See the Concepts Guide to learn more about how the triple quadrupole mass
spectrometer works and why the collision energy voltage is important. For
background information, see Chapter 3, Agilent Triple Quad MS and Sensitivity,
in the Concepts Guide. See the online Help for detailed information on how the
program works.
Each task is presented in a table with three columns:
Steps Use these general instructions to proceed on your own to explore the
program.
Detailed instructions Use these if you need help or prefer to use a step-by-step
learning process.
Comments Read these to learn tips and additional information about each
step in the exercise.
2 Agilent 7000 Series Triple Quad GC/MS Familiarization Guide

Before you begin
Prepare your system
Before you begin

Before you begin, you need to check that your system is ready. If you plan to
acquire data, you also need to set up the instrument.
Prepare your system

1 Check that:
The Data Acquisition, Qualitative Analysis, and Quantitative Analysis
programs are installed.
Your system uses an Agilent 7890 Series GC with split/splitless inlet and
automatic liquid sampler.
The 7000 Series Triple Quad GC/MS is configured.
The performance is verified.
The system is turned on.
The Agilent HP-5MS 5% Phenyl Methyl Siloxane: 30 m x 250 m x 0.25 m
column is installed.
2 Configure the GC for the Agilent HP-5MS column.
3 Copy the data files to your PC.
4 Copy the data files in the Benzodiazepine\Data folder on your Data
Acquisition installation disk to any location on your hard disk. This folder
contains the data files needed for this exercise.
Do not re-use the Benzodiazepine data files already on your system unless you
know that you copied them from the originals on the disk and you are the only one
using them. Do not use these sample data files to look at sample information or
print a report.
Prepare the samples required for data acquisition

If you do not intend to acquire data but want to learn how to use the Qualitative
and Quantitative Analysis programs for method development, you can skip this
section. You can learn how to use the Qualitative and Quantitative Analysis
programs with the Benzodiazepine data files shipped with the system.
Materials required for sample preparation:
A 1-mL ampoule of a Benzodiazepine mix sample, Agilent part number
B-033.
Acetonitrile for sample dilution
Sample vials
Agilent 7000 Series Triple Quad GC/MS Familiarization Guide 3

Before you begin
Prepare the samples required for data acquisition
1 Prepare the Qualitative Analysis samples.

a Add a quantity from the Benzodiazepine ampoule to acetonitrile in a glass
vial or tube and dilute twice so that the final concentration is 10 ng/L.
b Prepare 10 ALS sample vials using the solution obtained above.
2 Prepare the Quantitative Analysis calibration samples.
a Add a quantity from the Benzodiazepine ampoule to acetonitrile in an
Eppendorf vial and dilute to final concentrations of 1, 0.5, 0.25, 0.125, and
0.0625 ng/L.
b Prepare 5 ALS sample vials for the five concentration level solutions
obtained above. Label the vials BenzoL1 through BenzoL5.
3 Prepare the Quantitative Analysis unknown samples.
a Add a quantity from the Benzodiazepine ampoule to acetonitrile in a glass
vial or tube and dilute to final concentrations of 0.4, 0.2, and 0.1 ng/L.
b Prepare 3 ALS sample vials for the solutions obtained above. Label the vials
BenzoSample01 through BenzoSample03.
Before proceeding with this exercise, autotune the instrument. See the Operators
Manual or online Help for instructions on tuning the instrument.

Exercise Develop an acquisition method for the 7000 Series
Task 1. Set the inlet and injection parameters

l
Steps Detailed instructions Comments
1 Set up the inlet, injection source, a Double-click the Data Acquisition icon The Data Acquisition window
and enable the 7000 Series. on the windows desktop. shown in Figure 1 is displayed.
b Click the Inlet and Injection The Inlet and Injection Parameters
Parameters icon. dialog box shown in Figure 2 is
c Select GC for the sample inlet and the displayed.
installed ALS for the injection source.
d Select the Use MS check box.
Edit Sequence QQQ Method Editor

GC Edit Parameters
Inlet and Injection Parameters
Figure 1 Agilent MassHunter Workstation Software Data Acquisition window

Figure 2 Inlet and Injection Parameters

Task 2. Enter GC acquisition parameters

In this exercise, you enter the GC conditions for the analysis.
l
2 Enter GC parameters appropriate a Click the GC Edit Parameters icon The GC edit parameters window
for the Benzodiazepine mix. See (Figure 1). shown in Figure 3 on page 8 is
Table 1. b Select the CFT Settings icon then displayed.
select column 1 in the Description With the window selected, mouse
column. over the icons to identify the icon
c Select control mode On and then from the tool tip.
select Flow mode. Enter 1.2 mL/min
for the initial Value and the Post Run
value.
d Select the collision cell N2 EPC The collision cell gas flow is off for
module in the Description column and this method.
clear control On.
e Select the collision cell He EPC
module in the Description column and
clear control On.
f Select the Inlets icon then the SSL tab
and enter the inlet parameters listed in
Table 1.
g Select the Oven icon and enter the
oven parameters listed in Table 1.
h Select the ALS icon then the Front If your ALS is attached to the Back
Injector tab and enter the injector Inlet select the Back Injector tab.
parameters listed in Table 1.
i Select the Aux Heaters icon, enable,
and set the temperature to 134 C.
j Select the OK button. The GC parameters are downloaded
to the GC and the window closes.

Figure 3 GC Edit Parameters window
Table 1 GC parameters for data acquisition method
Parameter Value
Oven
Equilibration Time 0.5 min
Oven Program 100 C for 1 min, 25 C/min to 300 C, hold for 10 min
Run Time 19 min
Front SS Inlet He
Mode Splitless
Heater On 300 C

Parameter Value
Pressure On Value automatically set with CFT Setting column flow
Septum Purge Flow On 3 mL/min
Gas Saver On 20 mL/min after 3 min
Purge Flow to Split Vent 50 mL/min at 1 min
Thermal Aux 2 {MSD Transfer Line}
Heater On
Temperature 134 C
Column # 1 HP-5MS 5% Phenyl Methyl Siloxane: 30 m x 250 m x 0.25 m
In Front SS Inlet He
Out Vacuum
(Initial) 100 C
Flow 1.2 mL/min
Flow Program Off
Front Injector
Syringe Size 10 L
Injection Volume 1 L
Solvent A Washes (PreInj) 2
Solvent A Washes (PostInj) 4
Solvent A Volume 2 L
Solvent B Washes (PreInj) 2
Solvent B Washes (PostInj) 4
Solvent B Volume Max
Sample Washes 1
Sample Wash Volume 5 L
Sample Pumps 5
Dwell Time (PreInj) 0 min

Parameter Value
Dwell Time (PostInj) 0 min
Solvent Wash Draw Speed 300 L/min
Solvent Wash Dispense Speed 6000 L/min
Sample Wash Draw Speed 300 L/min
Sample Wash Dispense Speed 6000 L/min
Injection Dispense Speed 6000 L/min
Viscosity Delay 7 sec
Sample Depth Disabled
Collision cell EPC Module
Nitrogen Off
Helium Off

Task 3. Create acquisition method for finding precursor ions

In this exercise, you start with the GC parameters entered to the method in Task 2,
then enter the 7000 Series parameters for scanning the precursor ions and save to
the method.
3 Enter MS parameters appropriate a Click the QQQ Method Editor icon The QQQ Method Editor window
for the Benzodiazepine mix and (Figure 1). shown in Figure 4 opens.
save the method as b Set the Source temperature to 250 C,
iiipaul1_MS_Scan.M, where iii set the Electron energy to Tune
are your initials. Setting, the Solvent delay to 4
minutes, and the Detector setting to
Delta EMV.
c Set the Time Filtering to a Peak width
of 0.7 seconds.
d In the Time segment section, enter a The 7000 Series starts collecting
start Time of 3.0 and select a Scan data at 4 minutes due to the
Type of MS1Scan from the drop-down Solvent delay setting.
list. Enter 0 for Delta EMV and select
Data stored.
e In the Scan segments section, enter
Scan for the Segment name, 50 for the
Start mass, 450 for the End Mass, and
500 for the Scan time.
f In the Scan parameters section, enter
a Step size of 0.1 amu and a Threshold
of 100.
g Click OK to close the window.
h From the main window select
Method > Save Method As and save
the method as iiipaul1_MS_Scan.M,
where iii are your initials.

Figure 4 QQQ Method Editor

Task 4. Acquire precursor ion scan data (Optional)

In this task, you acquire the scan data using the method developed in the previous
tasks. This task is optional because you can perform the next task with an example
data file that comes with the program. If you prefer, you can create your own data
file as described in this task.
run
4 Acquire data (optional). a Click the Start Run (green arrow) icon. The Start Run dialog box shown in
Name the data file b In the Data Path enter the directory to Figure 5 is displayed.
iiifirst_trial3_scan.D, where iii save the data file that is acquired by
are your initials. this run.
Designate a directory path to c In the Front Inlet section, enter
hold your data files and method. iiifirst_trial3_scan.D for the Data File
Name, where iii are your initials.
d Enter the Vial location number in the
auto sampler tray.
e In the Method Sections to Run
section, select Data Acquisition.
f Click the OK and Run Method button. The method is sent to the GC and
the 7000 Series. When the
instruments are ready the sample is
injected and the data is collected
and sent to the data directory
specified.

Figure 5 Start Run dialog box

Task 5. Determine precursor ion masses

In this exercise, you determine the precursor ions for each of the compounds in
the acquired data file.
5 Open the acquired data file. a Double-click the Qualitative Analysis When you open a data file, the
In the Qualitative Analysis icon. Load result data (lower left corner)
program, open either the The program displays the Open Data check box is grayed out if the result
example file, File dialog box. data was not saved. If you see the
first_trial3_scan.D, or the data check box selected, this means that
file you created in Task 1. Set the data file(s) already contains
the inlet and injection results. Clear this check box
parameters on page 5. before opening the file.
Before you begin, make sure that all

previous settings are returned to their
default values:
Restore default layouts
Click View > Window Layouts
> Restore Default Layout.
Make sure the method is
default.m. (see title bar)
Click Method > Open.
Select default.m, and click
Open.
Return display options to default
settings.
Click Tools > Plot Display
Options...
Click Default, and then OK.


b Do one of the following: The figure below shows the default
Select the example data file layout. This is what you want to
first_trial3_scan.D, and click Open. see.
Select the data file you created in The Qualitative Analysis program
Task 4. Acquire precursor ion scan displays a newly opened data file
data (Optional) on page 13, and with the same layout and display
click Open. settings used for the previous data
By default, the system displays the file. Therefore, you MUST make
Total Ion Chromatogram (TIC). sure to return to the default
settings for this exercise.


Determine precursor ion masses c In the Chromatogram Results window,
for all eight peaks. the Range Select icon in the toolbar
You have determined them is selected.
correctly if you find the values d Click the left mouse button and drag
are similar to those shown in the cursor across the first peak at RT
this table. of about 9.0, to produce a shaded
Close the data file after finding region, as shown below.
the precursor ion masses. e Right-click inside the shaded area, and An averaged spectrum from the
select Extract MS Spectrum from the highlighted area inside the peak is
shortcut menu. displayed in the MS Spectrum
PEAK Compound RT m/z f Repeat step d through step e for the Results window.
1 Oxazepam 9.0 239.1 other compounds. The precursor mass of the first
2 Lorazepam 9.3 274.1 The precursor ion masses should compound, Oxazepam, is
3 Diazepam 9.4 283.2 match those in the table in step 2. determined to be m/z 239.1.
4 Temazepam 10.0 271.1 g Click File > Close Data File.
5 Flunitrazepam 10.1 312.2 h When asked if you want to save the
6 Nitrazepam 10.9 253.1 results, click No.
7 Clonazepam 11.4 314.1
8 Alprazolam 12.1 308.2

Task 6. Create acquisition methods for finding product ion masses

In this part of the method development, we will use four collision energies to
determine the best product ion to use for Multiple Reaction Monitoring (MRMs).
We start with the method previously saved in Task 3. Create acquisition method
for finding precursor ions on page 11 and change the 7000 Series part of the
acquisition method to fragment the previously identified precursor ions and scan
for product ions at four different collision energies.
6 Enter MS parameters appropriate a From the Data Acquisition workstation This method was previously saved
for finding the Oxazepam product open the iiipaul1_MS_Scan.M in Task 3 and the GC acquisition
ion and save the method as method, where iii are your initials. parameters provide excellent
iiipaul1_Oxazepam_prod_ion_Sc b Click the QQQ Method Editor icon. compound separation.
an2.M, where iii are your initials. c In the Time segment section, enter a The QQQ Method Editor window
start Time of 3.0, select a Scan Type of opens.
Create additional methods for the Product Ion from the drop-down list,
other 7 compounds using the data enter 650 for the Delta EMV, and
from the previous task. select Data stored.
d In the Scan segments section, enter
Oxazepam for the Segment name,
239.1 for the Precursor ion, 50 for MS2
from, 275 for MS2 to, 300 for Scan
time (ms), and 5 for the Collision
energy.
e Set the MS1 resolution to Wide.
f In the Scan parameters section, enter
a Step size of 0.1 amu and select
Profile data.
g In the Scan segments section, A new scan segment with all
right-click a cell and select New Scan values from the first scan segment
Segment from the context menu. is created.
h Repeat the above step three times. A maximum of four scan segments
is supported on the 7000 Series.

i Change the Collision energy to 15 for A total of four scan segments, each
the second scan segment. with a different collision energy, is
j Change the Collision energy to 25 for required for optimizing product ion
the third scan segment. sensitivity and selectivity.
k Change the Collision energy to 35 for
the fourth scan segment.
l Click OK to close the window.
m From the main window select
the method as
iiipaul1_Oxazepam_prod_ion_Scan2.
M, where iii are your initials.
n Click the QQQ Method Editor icon.
o For each compound shown in Table 2, Eight compound specific methods
create a new method by repeating the are created.
above steps substituting the values
from Table 2 and saving each method
to the table named method.
Table 2 Required methods and changed values for finding product ions
Method Segment Name Precursor MS2 MS2 to Scan Collision

Ion from time energy
iiipaul1_Oxazepam_prod_ion_Scan2.M Oxazepam 239.1 50 275 300 5, 15, 25, 35
iiipaul1_Lorazepam_prod_ion_Scan1.M Lorazepam 274.1 50 280 300 5, 15, 25, 35
iiipaul1_diazepam_prod_ion_Scan1.M Diazepam 283.1 50 290 300 5, 15, 25, 35
iiipaul1_flunitrazepam_prod_ion_Scan1.M Flunitrazepam 312.2 50 315 300 5, 15, 25, 35
iiipaul1_temzepam_prod_ion_Scan1.M Temazepam 271.1 50 280 300 5, 15, 25, 35
iiipaul1_nitrazepam_prod_ion_Scan1.M Nitrazepam 253.1 50 270 300 5, 15, 25, 35
iiipaul1_clonazepam_prod_ion_Scan1.M Clonazepam 314.1 50 320 300 5, 15, 25, 35
iiipaul1_alprazelam_prod_ion_Scan1.M Alprazolam 308.2 50 320 300 5, 15, 25, 35

Figure 6 Oxazepam 7000 Series acquisition method for determining the product ion

Task 7. Acquire scan data for finding product ions (Optional)

In this task, you create a sequence to acquire data for finding the product ions of
the Benzodiazepine compounds using the eight compound specific methods
developed in the previous task.
7 Set up and run a sequence a Click the Sequence Edit icon to display This step is optional because you
(optional). the Sequence Table. can use the six example data files
Name the data files b Use the drop-down list next to the in the next step.
iiixx_prod_ion_scan1.d, where New Sample(s) button in the The table now contains 8 sample
iii are your initials and xx is the Sequence Table toolbar and select 5 lines.
compound name. Samples.
c Enter a sample Name, Vial ALS
location number, Method File name,
and Data File name for each sample as
shown in Figure 7.
d Use the toolbars Fill down drop-down
list to copy the Type column Sample
value and the Dil. column 1 value to all
samples.
e Click the OK button.
f Select Sequence > Save Sequence As
to save the sequence as
BenzoPI.Sequence.xml.
g Place eight sample vials in the
specified location of the ALS tray.
h Click the Sequence Run icon to display
the Start Sequence dialog box.
i If needed, add a sequence Comment,
change the Data File Directory and
click the Run Sequence button.

Figure 7 Sequence Table values
Figure 8 Start Sequence dialog box

Task 8. Determine the product ions

In this exercise, you determine the product ion for each compound in the
compound specific acquired data file.
8 Open the acquired data files in the a Double-click the Qualitative Analysis When you open a data file, the
Qualitative Analysis program. icon. Load result data (lower left corner)
The program displays the Open Data check box is grayed out if result
File dialog box. data was not saved. If you see the
check box selected, this means that
the data file(s) already contains
results. Clear this check box
before opening the file.
b Do one of the following: Use the CTRL key to select multiple

Select the eight example data files data files.
and click Open.
Select all the data files you
acquired in Task 7. Acquire scan
data for finding product ions
(Optional) on page 21, and click
Open.
c In the Data Navigator, clear the check Only a single acquisition file at a
boxes for all compounds except time is displayed.
Oxazepam.
d From the MS Spectra Results window A maximum of 5 spectrum panes
toolbar, enter 5 from the drop-down can be displayed.
menu.


e In the Chromatogram Results window,
the Range Select icon in the toolbar
is selected.
f Click the left mouse button and drag For Oxazepam this peak is at about
the cursor across the prominent peak 9 minutes.
corresponding to the RT of this
compound, to produce a shaded
region.
g Right-click inside the shaded area, and An averaged spectrum from the
select Extract MS Spectrum from the highlighted area inside the peak is
shortcut menu. The spectrum for this displayed in the MS Spectrum
peak is shown in Figure 9. Results window for each of the four
collision energy scan segments in
the acquisition method.
Figure 9 Extracted spectra for 4 collision energy scans showing product ion selected

Determine product ion masses for h The product ion for the first compound, Examine each spectrum to find the
all eight peaks. Oxazepam, is determined to be m/z most selective ion. Look for the
You have determined them 177.1 at CID 25. highest count of a mass closest to
correctly if you find the values i Clear the check box for the current the precursor ion with minimum
are similar to those shown in data file in the Data Navigator. interference from adjacent ions.
Table 3. j Select the check box for the next data
Close the data file after finding file. Here the best ion selection has a
the precursor ion masses. k Repeat step f through step j for the count of 100 compared to other
other compounds. ions with a count of 25 maximum in
The product ion masses should match any other spectrum.
those in Table 3.
l Click File > Close Data File.
m When asked if you want to save the
results, click No.
Table 3 Product Ions found for the Benzodiazepine compounds
Compound Data File Precursor Ion Product Ion Collision Energy
Oxazepam Oxazepam_prod_ion_scan2.d 239.1 177.1 25
Lorazepam Lorazepam_prod_ion_scan1.d 274.1 239.1 15
Diazepam Diazepam_prod_ion_scan1.d 283.1 238.1 25
Flunitrazepam Flunitrazepam_prod_ion_scan1.d 312.2 266.1 25
Temazepam Temazepam_prod_ion_scan1.d 274.1 77.1 35
Nitrazepam Nitrazepam_prod_ion_scan1.d 253.1 152.1 25
Clonazepam Clonazepam_prod_ion_scan1.d 314.1 268.2 25
Alprazolam Alprazolam_prod_ion_scan1.d 308.2 279.2 15

Task 9. Create an MRM method

In this exercise, you create an MRM method that finds any Benzo compound in a
sample.
9 Open the Data Acquisition program a From the Data Acquisition workstation The GC acquisition parameters
and create an MRM method using open the iiipaul1_MS_Scan.M from this method are used
the data from Table 3 on page 25. method, where iii are your initials. unchanged. The 7000 Series
b Click the QQQ Method Editor icon. method parameters are edited in
c In the Time segment section, enter a this procedure to create the MRM
start Time of 3.0, select a Scan Type of method.
MRM from the drop-down list, enter
650 for the Delta EMV, and select Data
stored.
d In the Scan segments section, enter
Wide for the MS1 Resolution and MS2
Resolution, and 50 for the Dwell.
e In the Scan segments section,
right-click a cell and select New Scan
Segment from the context menu.
f Repeat the above step seven times.
g Fill in the rest of the Scan Segments
section to match Figure 10 on page 27.
h Click OK to close the window.
i From the main window select
the method as
iiipaul1_benzo_mrm_1.M, where iii
are your initials.

Figure 10 Scan section completed for an MRM method

Exercise Create a quantitative analysis method
Task 10. Acquire MRM data (Optional)

In this exercise, you create a sequence to acquire calibration data used for the
quantitative analysis of MRM-acquired samples containing Benzo compounds.
Additionally there are 3 samples that contain concentrations of the Benzo
compounds to demonstrate the quantitation of unknown samples. The data
acquisition portion of the program is optional for the Benzo calibration samples
since example data files are included on the program disk.

10 Open the Data Acquisition program a From the Data Acquisition workstation This method was created in Task
and use the MRM method created load the iiipaul1_benzo_mrm_1.M 9. Create an MRM method on
in the last task to acquire method. page 26.
calibration data. This data is b Click the Edit Sequence icon to display
required to create a quantitative the Sequence Table.
analysis method in the Quantitative c Add or Delete sample entries so that To delete consecutive sample
Analysis program. the Sequence Table contains five entries, click in the left sample
sample lines. number column of the first sample
d For the first sample enter BenzoLevel1 to delete, hold down the Shift key
for the Name, enter the ALS vial and click the last sample number
location for the Vial, select column in the table. With the
Benzo_MRM.M for the Method File, samples to delete highlighted,
enter paul1_benzo_L1_19Nov08.D for press the Delete key on your
the Data File, select Cal from the computer.
drop-down list for the Type, enter L1 The Vial location incremented in
for the Level, and enter 1 for the this way assumes that the vials are
Dilution. See Figure 11 on page 30. placed in consecutive locations in
e Use the Fill Increment drop-down in the sampler tray.
the Sequence Table toolbar to copy
and increment the values listed for the
first line sample values for Name, Vial,
and Level to the other four samples.
f Use the Fill drop-down in the
Sequence Table toolbar to copy the
values listed for the first line sample
values for Method File, Data File,
Type, and Dilution to the other four
samples.
g Change _L1_ part of the Data File
name to follow the pattern shown in
Figure 11 on page 30.
h Add three additional lines to the
sample table and fill in the values for
these samples so that the completed
table resembles Figure 11 on page 30.
i Click OK to close the window.

j Select Sequence > Save Sequence As

to save the sequence as
BenzoCalibration.Sequence.xml.
k Place the five sample vials containing The remaining steps are optional.
known concentrations and the 3 These sample vials are prepared in
samples containing the unknown Prepare the samples required for
concentrations in the specified data acquisition on page 3.
locations of the ALS tray.
l Click the Sequence Run icon to display
the Start Sequence dialog box.
m If needed, add a sequence Comment,
change the Data File Directory and
click the Run Sequence button.
Figure 11 Sequence for Benzo calibration samples and unknowns

Task 11. Create a quantitative analysis batch

In this exercise, you create a batch that is used to create a quantitative method
using data acquired from the five calibration samples ran in the last task.
11 Open the Quantitative Analysis a Double-click the QQQ Quantitative The Quantitative Analysis
program and create a batch to Analysis icon on your windows Workstation opens.
analyze data. desktop.
Name the batch and assign a batch b From the main menu, select File > This is the directory selected for the
directory. New batch, use the Look In data files in Task 10. Acquire MRM
drop-down list to navigate to the data (Optional) on page 28. If you
directory containing the 5 calibration skipped this data acquisition task
files, and enter the batch name you can substitute the 5 sample
BenzoCalibrationData and click the data files included on the program
Open button. disk. Just copy these sample data
files to this directory.
Add samples to the batch. c From the main menu, select File > Add The 5 calibration files are need to
samples and select the 5 data files create the data analysis calibration
paul1_benzo_L1_19Nov08.D through curve and one of these files is used
paul1_benzo_L5_19Nov08.D. for extracting MRM data for the
method.
The Type for all 5 samples is Cal
and the Level should range from L1
to L5. The Type and Level are taken
from the acquisition sequence
Table 11 on page 30.


d Click the OK button. The Batch Table The batch now requires a data
now contains the 5 samples. analysis method to automatically
e If necessary, change the Type to Cal, analyze the sample.
sort on the Name column, and enter
the values shown in the Level column.
f If necessary, add the Dil and Amt
columns to the Sample section of the
Batch Table. Enter a value of 1 for all
Dil and Amt entries in these columns.
.

Task 12. Create an MRM quantitative analysis method

12 Create a Data Analysis method. a From the main menu, select Method > The sample data stored in the
New > New Method from Acquired acquisition method created in
MRM Data to display the New Task 9. Create an MRM method
Method from Acquired Data dialog on page 26 is used to automatically
box. fill in parts of the data analysis
method.
b Use the Look In drop-down list to The program uses this sample data
select the batch directory. Click the file to create much of the data
paul1_benzo_L5_19Nov08.D data file, analysis method, then presents the
then click the Open button. user with the method editor so that
the method can be finished
manually.


c In the Method Tasks pane, click Review the eight compounds that
Method Setup Tasks, then click MRM were originally included in the
Compound Setup to display the acquisition method.
Method Table.


Create a calibration table. d In the Method Tasks pane, click The Method Table columns change
Method Setup Tasks, then click to assist with entering the
Concentration Setup. concentration levels.
e In the Dil. High Conc. column for the The program uses the highest
first compound in the table enter concentration for a calibration
1000.0 then select 1:2 from the sample, the number of calibration
drop-down list for the Dil. Pattern levels, and a dilution pattern to
column. automatically calculate the
concentrations for the other levels.

f In the Method Table toolbar, enter L for The calibration table containing a
a Level Name Prefix, and 5 for the # of concentration entry for each of the
Levels then click the Create Level 5 calibration levels is shown below
button. for the first compound in the
method table.

Copy the calibration table to other g Select the first compound in the The Copy Calibration Levels To
quantifiers. Quantifier Table, right-click the dialog box is displayed. Use this to
compound and select Copy copy the calibration table from the
Calibration Levels To from the current compound to selected or all
shortcut menu. other compounds.
h Click the Select All button to select all This calibration table is copied to
compounds in the dialog box then click the Quantifier table of all eight
the OK button. compounds.


Calibration Curve Setup i In the Method Tasks pane, click The Method Table is displayed with
Method Setup Tasks, then click the CF, CF Origin, and CF Weight
Calibration Curve Setup. columns highlighted.
j In the Quantifier table CF column of The Benzo compounds in this
the first compound select Quadratic example will have a CF of
from the drop-down list. With this cell Quadratic, a CF Origin of Ignore,
selected, right-click and select Fill and a CF Weight of None.
Down from the short-cut menu.


Select an Integrator. k In the Method Tasks pane, click You can select from several
Advanced Tasks, then click integrators with MassHunter.
Integration Parameters Setup.
l In the Method Table pane, for any Use the MS-MS (GC) integrator
Quantifier click the Int. column entry which is optimized for MRM data
to display the Integration dialog box with GC use. This is a
and select MS-MS (GC) from the parameter-less integrator.
Integrator drop-down list.
m Click the Apply to All button to set the

This integrator is used for
Integrator for all quantifier peaks to
integrating all peaks.
this value.
n Click OK to close this dialog box.
Exit the method editor and save the o In the Method Tasks pane, click
method. Save/Exit, then click Exit.


Apply the data analysis method to p Click the Yes button. The method is applied to all
the batch. samples in the Batch Table.
q Click the Analyze Batch button to Selecting Analyze Batch takes the
quantitate all samples in the batch. compound responses in each
calibration sample and uses them
to create the CurveFit equations for
each compound.
Review the calibration curve for r In the calibration curve pane, In the CurveFit table, click a filter
each compound. right-click and select Curve Fit icon next to the column name.
Assistant from the short-cut menu. Clicking the Type filter icon and
s In the CurveFit table, select a line with selecting Quadratic allows only
a Type of Quadratic, Ignore the Origin, quadratic equations for this
and use a maximum of 0 for the # of compounds calibration curve. Try
Disabled Points. this for the other criteria.


t Review the curve fit for different These CurveFits were originally
equations by selecting a different line selected non-graphically in step j.
in the CurveFit table. If you find a
better CurveFit, select it and in the
graph area of the calibration curve,
right-click and select Accept Assistant
Curve from the shortcut menu.
u In the Batch Table toolbar, click the The Calibration Curve pane only
right arrow to select the next displays the calibration data for one
calibrated compound and repeat steps compound at a time.
q through s.
v Repeat the above step until all eight
calibrated compounds are reviewed
and corrected if necessary.
w Click the Analyze Batch button to This step is only necessary if you
quantitate all samples in the batch. changed any CurveFit equation
during the review above.
Save the Data Analysis method x From the main menu select Method >
. Edit to display the Method Editor.
y In the Method Tasks pane, click Save
/ Exit, then click Save As.
z Enter BenzoDA for the method name
and click the Save button.

Task 13. Quantitate a batch of unknown samples

In this exercise, you create a batch that is used to quantitate unknown Benzo
samples using the quantitative method created in the last task. Sample data files
are not provided for these unknown Benzo samples. These data files are only
obtained by completing Task 10. Acquire MRM data (Optional) on page 28.
13 Open the Quantitative Analysis a Double-click the QQQ Quantitative The Quantitative Analysis
program and create a batch to Analysis icon on your windows Workstation opens.
analyze data. desktop.
Name the batch and assign a batch b From the main menu, select File > This is the directory included on the
directory. New batch, use the Look In program disk. Just copy these
drop-down list to navigate to the sample data files to this directory.
directory containing the 3 Benzo
sample files, and enter the batch name
BenzoSamples031709 and click the
Open button.
Add samples to the batch. c From the main menu, select File > Add
samples and select the samples
BenzoSample01.D through
BenzoSample03.D.
d Click the OK button. The Batch Table The batch now requires a data
now contains the 3 unknown samples. analysis method to automatically
. e If necessary, change the Type to analyze the sample.
Sample.
f If necessary, add the Dil and Amt
columns to the Sample section of the
Batch Table. Enter a value of 1 for all
Dil and Amt entries in these columns.
g In the main menu select Method >

Open > Open and Apply from existing
file to display the Open Method File
dialog box.
h Enter the BenzoDA.quantmethod.xml The method is applied to the batch.
for the File name and click the Open
button.
i Click the Analyze Batch button to
quantitate all samples in the batch.
j Review the sample results for each Print one or more reports.
compound in the method.


www.agilent.com
Agilent Technologies, Inc. 2013

Third Edition, October 2013
G6845-90001
Agilent Technologies

G6845-90001 QQQ Familiarizsation

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Agilent MassHunter Workstation

Software Data Acquisition for

2 Agilent 7000 Series Triple Quad GC/MS Familiarization Guide

Before you begin

Prepare your system

Prepare the samples required for data acquisition

Agilent 7000 Series Triple Quad GC/MS Familiarization Guide 3

1 Prepare the Qualitative Analysis samples.

4 Agilent 7000 Series Triple Quad GC/MS Familiarization Guide

Exercise Develop an acquisition method for the 7000 Series

Task 1. Set the inlet and injection parameters

Steps Detailed instructions Comments

Edit Sequence QQQ Method Editor

Figure 1 Agilent MassHunter Workstation Software Data Acquisition window

Agilent 7000 Series Triple Quad GC/MS Familiarization Guide 5

Figure 2 Inlet and Injection Parameters

6 Agilent 7000 Series Triple Quad GC/MS Familiarization Guide

Task 2. Enter GC acquisition parameters

Steps Detailed instructions Comments

Agilent 7000 Series Triple Quad GC/MS Familiarization Guide 7

Figure 3 GC Edit Parameters window

Table 1 GC parameters for data acquisition method

Equilibration Time 0.5 min

Run Time 19 min

8 Agilent 7000 Series Triple Quad GC/MS Familiarization Guide

Pressure On Value automatically set with CFT Setting column flow

Septum Purge Flow On 3 mL/min

Gas Saver On 20 mL/min after 3 min

Purge Flow to Split Vent 50 mL/min at 1 min

Thermal Aux 2 {MSD Transfer Line}

Column # 1 HP-5MS 5% Phenyl Methyl Siloxane: 30 m x 250 m x 0.25 m

Flow 1.2 mL/min

Flow Program Off

Solvent A Washes (PreInj) 2

Solvent A Washes (PostInj) 4

Solvent B Washes (PreInj) 2

Solvent B Washes (PostInj) 4

Solvent B Volume Max

Sample Wash Volume 5 L

Dwell Time (PreInj) 0 min

Agilent 7000 Series Triple Quad GC/MS Familiarization Guide 9

Dwell Time (PostInj) 0 min

Solvent Wash Draw Speed 300 L/min

Solvent Wash Dispense Speed 6000 L/min

Sample Wash Draw Speed 300 L/min

Sample Wash Dispense Speed 6000 L/min

Injection Dispense Speed 6000 L/min

Viscosity Delay 7 sec

Sample Depth Disabled

Collision cell EPC Module

10 Agilent 7000 Series Triple Quad GC/MS Familiarization Guide

Task 3. Create acquisition method for finding precursor ions

Steps Detailed instructions Comments

Agilent 7000 Series Triple Quad GC/MS Familiarization Guide 11

Figure 4 QQQ Method Editor

12 Agilent 7000 Series Triple Quad GC/MS Familiarization Guide

Task 4. Acquire precursor ion scan data (Optional)

Steps Detailed instructions Comments

Agilent 7000 Series Triple Quad GC/MS Familiarization Guide 13

Figure 5 Start Run dialog box

14 Agilent 7000 Series Triple Quad GC/MS Familiarization Guide

Task 5. Determine precursor ion masses

Steps Detailed instructions Comments

Before you begin, make sure that all